The mass production of recombinant molecules of commercial value is a technical area of increasing complexity and interest. Many different organisms have been considered as hosts for foreign protein expression including single-cell organisms such as bacteria and yeast, cell cultures of animals, fungi and plants, and whole organisms such as plants, insects, fungi and transgenic animals. In general, each particular organism has unique characteristics that may offer advantages for production of specific proteins of interest. Alternatively the specificity of certain protein production platforms may limit utility for widespread applications. Thus, numerous molecular farming systems have been developed as a means to produce proteins of commercial interest.
Of particular interest to the subject matter of the present invention is the expression of heterologous proteins in plant cells. Numerous foreign proteins have been expressed in whole plants and selected plant organs. Plants can offer a highly effective and economical means to produce recombinant proteins as they can be grown on a large scale with modest cost inputs and most commercially important species can now be transformed.
In order to optimize protein production and recovery, a number of factors need to be considered. These include the levels of recombinant protein production, the temporal aspects of recombinant protein production, and the stability of the final product within the plant cell. The level of protein production must be sufficient to allow accumulation of the product in quantities that are commercially valuable and can be conveniently isolated. In many instances, it may be desired that the temporal expression of the product coincide with the period when the crop is harvested or collected. In addition, it may be required that the protein stably accumulate to appreciable levels, or be induced to quickly accumulate to appreciable levels if the product is intrinsically unstable.
The production of heterologous proteins in plants has been achieved using a variety of approaches. U.S. Pat. Nos. 6,650,307, 5,716,802, 5,763,748 disclose recombinant protein production using transcriptional fusions to a constitutive plant promoter. Production of heterologous proteins in seed (U.S. Pat. Nos. 5,504,200; 5,530,194; 6,905,186; 5,792,922; 5,948,682), fruit (U.S. Pat. Nos. 6,783,394; 4,943,674) or storage organs such as tubers (U.S. Pat. Nos. 5,436,393, 5,723,757) have also been described.
A disadvantage of constitutive expression systems is that constitutive expression of a protein may lead to toxic effects with regards to plant growth. Furthermore, it is difficult to predict what interactions a foreign protein may have with other plant proteins, such as enzymes or receptors, plant membranes, such as those of the endoplasmic reticulum, Golgi apparatus, vacuole and plasmalemma, or the host of other molecules critical to the growth and development of the plant. Another potential disadvantage of a constitutive or non-inducible promoter is the metabolic cost of synthesizing the transgenic protein in all tissues at all stages of growth. If the only tissue to be harvested is the leaves, for example, it is inefficient and wasteful for the plant to produce the foreign protein in other tissues. Alternatively, if the transgene encoded protein is labile or unstable, then production of the protein, constitutively, throughout the growth of the plant is inefficient.
Inducible systems allow the expression of an introduced gene to take place at a desired time in the development of a plant, under specific circumstances or in specific tissues. For example, leaf-specific promoters or promoters induced in the leaves by some treatment would restrict synthesis to only the harvested tissue. In addition, an induced foreign gene is potentially less likely to undergo gene silencing than a transgene controlled by a constitutive or tissue specific promoter. Furthermore, inducible transgene systems offer a method of biological containment since the foreign protein is not present in the crop until the application of the inducing treatment, at which time the crop is harvested. Containment of a protein produced from a foreign gene is as, or more, important than containment of the gene as the protein is the biologically active component.
Gene expression in response to plant wounding is another potential source of an inducible system. U.S. Pat. Nos. 5,689,056, 5,670,349, 5,929,304, and U.S. Pat. No. 5,777,200 disclose the use of regulatory elements from wound inducible genes for the induction of heterologous protein synthesis in plants. However, the value of these wound-inducible promoters may be limited since wounding of the plant also induce other genes, such as proteases, that can negatively impact the production of the recombinant protein. It is also not clear that these regulatory elements provide sufficient levels of expression to cause accumulation of the recombinant protein to substantial levels, especially when the response is localized to the site of wounding (e.g. HMG2 promoter, U.S. Pat. No. 5,689,056). Although the expression levels with such promoters can be enhanced by applying more extensive wounding treatments or chemical inducers such as methyl jasmonate, this entails additional costs.
Thus, although promoters involved in inducible systems can provide powerful tools for control of transgenes in plants, many obstacles are faced in utilizing these regulatory elements. Inducible promoter systems must enable the precise timing and location of expression of such transgenes in order to be commercially useful. In this regard, regulatory elements that can be induced under precise conditions amenable to cultivation practices are desired. More particularly, there is a need for regulatory elements that are induced, specifically, during harvesting conditions.
Volenec et al. (“Molecular analysis of alfalfa root vegetative storage proteins” pp59-73 in Molecular and Cellular Technologies for Forage Improvement, CSSA Spec Publ. No. 26, 1998) have characterized the changes that ensue in root tissue following harvest and shoot regrowth of alfalfa (Medicago sativa L.). However, no specific regulatory elements were identified or characterized in any manner.
Ferullo et al (Crop. Sci. 1996 36, 1011-1016) disclose proteins that are specific to harvesting conditions of alfalfa. However the structure or function of these proteins was not characterized and is unknown; moreover, there is no indication of the nature of the genes expressed in harvested shoot tissue of alfalfa during harvesting. Furthermore, there is no suggestion as to the use of regulatory elements associated with these genes for induction of heterologous gene expression in plants in a harvest-inducible manner.
Coupe et al. (WO 00/31251) disclose the characterization of a promoter from asparagine synthetase and its use in post harvest gene expression.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.